ABSTRACT Non-alcoholic fatty liver disease (NAFLD) is emerging as one of the most prevalent chronic liver diseases in the world, with non-alcoholic steatohepatitis (NASH) being an extreme form of NAFLD. Our team proposes to investigate the novel concept that intestine-selective FXR antagonism has the potential to prevent or reverse NAFLD/NASH. FXR is the major bile acid sensor in the body and serves to mediate the dialog between the liver and small intestine regarding bile acid levels, bile acid synthesis, transport, and enterohepatic circulation, and also regulates lipid and glucose levels in the liver. As such, FXR was identified in human trials as a promising target for the prevention and/or amelioration of many metabolic diseases, including NAFLD/NASH. We have recently established intestinal FXR as a major regulator of diet-induced obesity and NAFLD, particularly through antagonism of the receptor with the conjugated bile acid tauro-?-muricholic acid (T?MCA). We reported that tempol, a potent antioxidant, inhibited the FXR signaling pathway, due to accumulation of intestinal T?MCA, an antagonist of FXR, resulting from reduced activity of the Lactobacillus-associated bile salt hydroxylase enzyme. We further developed glycine-?-muricholic acid (Gly?MCA), a potent, intestine-selective FXR antagonist that similarly prevents or reversed NAFLD in diet-induced NAFLD models or genetic models, suggesting intestinal FXR as a promising therapeutic target for NAFLD. These observations have led to the novel central hypothesis: Antagonism of intestinal FXR prevents the development of NAFLD In this proposal we plan to address two fundamental gaps in knowledge with respect to FXR antagonism and NAFLD. First, we will identify using a combination of in vitro and in vivo approaches what chemical features define an FXR antagonist and develop a better understanding for how these modifications influence the absorption, distribution, metabolism, and excretion (ADME) of the antagonist and its pharmacodynamic properties. Second, through the innovative use of cutting-edge techniques?including 16S rRNA gene sequencing, metagenomics, metatranscriptomics, and metabolomics?and unique mouse models, we plan to identify how FXR signaling and the gut microbiota are linked in NAFLD pathogenesis. This combination of approaches will allow us to identify new therapeutic FXR antagonists and to accurately assess how these compounds influence downstream FXR signaling pathways with the ultimate goal of developing new NAFLD therapies.